Total Hydroxyproline Research Articles

The urinary excretion of collagen degradation products by quadriplegic patients and during weightlessness

Urinary total hydroxyproline, peptide-bound hydroxyproline, hydroxylysine glycosides, and calcium were measured in three healthy individuals subjected to weightlessness for eighty-four days and in three young quadriplegic patients. The former group had significant calciuria but no evidence of collagen degradation. The latter group, however, had conspicuous calciuria and also excreted large amounts of collagen breakdown products. This documented degradation of matrix may be related to the continuous calciuria, osteoporosis, and cutaneous dystrophic changes occurring in quadriplegics.

Biochemical measurements in Paget's disease of bone

In a group of eighty-three patients with untreated Paget's disease of bone, plasma alkaline phosphatase activity (AP), plasma non-protein-bound hydroxyproline concentration (PHP) and urinary excretion rate of total hydroxyproline (THP) were closely correlated with each other but not with fasting plasma concentrations of calcium or inorganic phosphate. Probit plots of AP and THP showed log-normal distributions overlapping the normal ranges.

Total hydroxyproline in urine of 4 to 6 year-old children--an investigation on its relationship to growth and nutrition.

We analysed over a period of 30-32 days the daily total hydroxyproline and creatinine excretine in urine from 9 healthy, normally fed 4 to 6 year-old children (2 girls, 7 boys). The average urinary hydroxyproline excretion was 45.6 mg/24 hr, with a coefficient of variation of 25.6%. Urinary hydroxyproline for individual children showed distinct differences from day to day, which were independent of urine volume. There were significant differences between the mean values for urine hydroxyproline in individual children, which were independent of age. The average creatinine in urine was 346/24 hr with a coefficient of variation of 17.7%. The hydroxyproline-index did not define the nutritional state of these normally developed children on a normal diet. Dietary hydroxyproline contributed 7.4% to the total urinary hydroxyproline in our investigation. There was a close correlation between urine hydroxyproline excretion and growth velocity in each child.

Effects of aldosterone on the urinary excretion of total and non-dialyzable hydroxyproline.

In order to explore the role of mineralocorticoids on collagen metabolism, the effects of aldosterone on the urinary excretion of total and non dialyzable hydroxyproline (HYPRO) was studied in rats. The administration of aldosterone in sesame oil, 75 microgram/100 g body weight to adrenalectomized rats maintained on 1% NaCl solution as drinking fluid and 1 mg of cortisone subcutaneously daily, provoked elevation of total and non dialyzable HYPRO in urine (P less than 0.001), when compared to similarly treated adrenalectomized rats receiving sesame oil but no aldosterone. Both groups showed a normal growth curve and had similar urinary excretion of creatinine. The effects of aldosterone are opposed to the known lowering effects of glucocorticoids on HYPRO excretion and may suggest an effect of aldosterone on collagen turnover. Alternatively, aldosterone may modify the metabolism or excretion of HYPRO in an opposite manner to that of glucocorticoids.

The influence of fluocortolone treatment on the collagen content in guinea pig skin.

Specific pathogen free guinea pigs were treated with varying dosis of fluocortolone for 15 days. The treated animals showed the same per cent weight gain as the controls. The total hydroxyproline content of the skin and the hydroxyproline content in different collagen fractions was the same in treated and untreated animals. Thus fluocortolone seems to have no specific effect on the synthesis or the breakdown of collagen in the guinea pig skin. Also the physical development of the animals, kept under defined conditions, failed to show a catabolic effect of of fluorcortolone.

Urinary hydroxyproline concentration in primary hyperparathyroidism with and without renal stones.

Eleven cases with histologically confirmed primary hyperparathyroidism have been studied. Although histologically, bone turnover increased in all but 1 patient, urinary hydroxyproline excretion and serum alkaline phosphatase in patients with renal stones were within the upper normal limits of slightly elevated (27.5 mg/24 h, concentration 19.5 microgram/ml, alkaline phosphatase 35.0 IU/l). On the contrary, 3 cases without renal stones exhibited an increased urinary hydroxyproline excretion (129 mg/24 h, concentration 95.6 microgram/ml) and elevated serum alkaline phosphatase (99.9IU/l). Serum total hydroxyproline was elevated in both groups (renal stones, 2.00 mg%; no renal stones, 3.16 mg%; p = 0.006). Hydroxyproline was determined under conditions of a very low proline diet and 1.5 liters of daily fluid intake. There were no statistically significant differences between serum calcium, phosphorus, and parathormone between urinary excretion of calcium and phosphorus. Creatinine clearance was within normal limits in all patients. The possible relevance of urinary hydroxyproline for stone formation is discussed.

Serum proline imino-peptidase activity in normal adult subjects and in patients with paget's disease of bone

The activity of proline imino-peptidase (EC 3.4.1.4) may provide a measure of collagen degradation, and a method for its estimation in human serum is described. The mean value of this enzyme activity in 48 normal adults of 4.7 mU/1 (S.D. 0.9) did not change with sex, age or dietary collagen. In 8 patients with liver disease associated with elevated levels of plasma alkaline phosphatase activity the mean value was normal (4.5 mU/1, S.D. 1.5). However, in 25 patients with untreated Paget's disease of bone, values were significantly increased (mean 7.4 mU/1; S.D. 2.8; p < 0.001) and were positively correlated with plasma alkaline phosphatase activity ( r = 0.75; p < 0.001) and with urine total hydroxyproline excretion ( r = 0.68; p < 0.001). In patients given disodium ethane-1-hydroxy-1, 1-diphosphonate for 3–6 months, serum proline imino-peptidase activity decreased to normal values.

Radioimmunoassay of procollagen in serum of patients with Paget's disease of bone.

A new radioimmunoassay for human procollagen showed that the sera of 46 of 50 untreated patients with Paget's disease of bone contained increased concentrations of procollagen protein as compared to normal adults. After therapy with disodium etidronate, all the elevated serum procollagen concentrations decreased significantly, falling to normal levels in 33 of 40 patients. The procollagen levels before and after treatment were coordinate with the values for urinary total hydroxyproline and serum alkaline phosphatase activity. The data show that the radioimmunoassay for procollagen is a dependable and useful adjunct to the study of Paget's disease of bone.

Parathyroid hormone and vertebral osteosclerosis in uremic patients

Six out of 30 patients with chronic renal failure showed osteosclerosis in the lateral radiograph of the lumbar spine. When two groups with a similar degree of renal impairment were compared, the patients with osteosclerosis were younger and had a significantly higher level of circulating PTH ( p < 0.05) and total hydroxyproline excretion ( p < 0.02), than patients with-out overt osteosclerosis. The metacarpal cortical thickness was significantly reduced in patients with vertebral osteosclerosis. The results suggest that in patients with chronic uremia endogenous hypersecretion of PTH is one of the most significant factors responsible for the development of osteosclerosis. The mineral released from other skeletal sites could be utilized in the mineralization of the newly formed trabecular bone without any external calcium gain.

THE VITAMIN D3 METABOLITE‐TYPE ACTIVITY OF SOLANUM MALACOXYLON

1. Administration of an aqueous extract of the dried leaves of Solanum malacoxylon (DLSM) to rats causes a rapid hyperphosphataemia and a decrease in plasma alkaline phosphatase activity; the two effects are typical of 1,25(OH)2D3, the hormonally active metabolite of vitamin D3. 2. DLSM, like both vitamin D3 and parathyroid hormone, increases plasma calcium and citrate levels in rats. The effect of DLSM in influencing plasma citrate, and the role of this important metabolite in mineral metabolism is discussed. 3. A decrease of plasma magnesium levels occurs in rats following treatment with DLSM. This decrease, which is associated with a renal loss of this cation, is remarkably similar to that produced by hypervitaminosis D3. 4. Prolonged administration of DLSM to vitamin D deficient rats causes a polyuria, hypercalciuria, hyperphosphaturia, hypermagnesuria, an increase in urinary total hydroxyproline, an increase in plasma total hexosamines, and a corresponding decrease in the bone total hexosamines. These effects, some of which can also be produced by hyperparathyroidism, or following the administration of parathyroid extract (PTE), large doses of vitamin D3, or 1,25(OH)2D3, suggest that DLSM, like the latter compounds, is capable of causing bone mineral mobilization, and the dissolution of bone organic matrix.

Relation of urinary total hydroxyproline: Creatinine ratio to height velocity in children with retarded growth.

The urinary total hydroxyproline:creatinine ratio was measured on samples collected from 18 boys and 11 girls referred to a children's hospital because of small stature. Any serious degree of growth hormone deficiency was excluded and each child's growth and development was recorded over the following year. The hypothesis that the average hydroxyproline:creatinine ratio on random urine samples may be used to predict height velocity was examined. Such a prediction would have been incorrect on 35% of occasions, and it is concluded that this investigation cannot be used reliability to predict growth in height; it is probably only useful with sequential studies of an individual child over short periods while monitoring therapy.

The removal of non-collagen components from newborn calf dermis with magnesium chloride solution.

1. Non-collagenous substances in newborn calf dermis were extracted with solutions of various concentrations of MgCl2. The total protein and hydroxyproline contents in MgCl2 extracts increased with increase in the concentration of MgCl2 in the solutions. In particular, steep increases of their contents were observed at concentrations of MgCl2 from 0.5 to 1.0 M. Total amounts of hydroxyproline in 1.0, 2.0, and 3.0 M MgCl2 extracts were equivalent to 40-50% of the hydroxyproline content in the whole connective tissue. Hexose and hexosamine contents of MgCl2 extracts increased with increase of the MgCl2 concentration. Hexuronic acid was hardly present in the residues after extractions with 0.5, 1.0, 2.0, and 3.0 M MgCl2. 2. Plasma proteins, hyaluronic acid, and dermatan sulfate were extracted at low concentrations of MgCl2. A non-collagenous protein and MgCl2-soluble collagen were extracted with 1.0, 2.0, and 3.0 M MgCl2 solutions. The disperson of collagen fibrils was observed in the residue extracted with 1.0 M MgCl2 solution by electron microscopy; the fibril structure of collagen was disordered by extraction with 2.0 and 3.0 M MgCl2. The results suggest that the dispersion and disorder of collagen fibrils lead to the release of a non-collagenous protein. Furthermore, it is suggested that the removal of hyaluronic acid and dermatan sulfate was not very effective for the solubilization of a large amount of collagen, but was suitable as a pretreatment to the extraction of a non-collagenous protein accompanied by the solubilization of a large amount of collagen. 3. The non-collagenous protein was purified by DEAE-cellulose column chromatography. Polyacrylamide gel electrophoresis of this protein at pH 8.5 showed a single band moving to the cathode. The non-collagenous protein contained 3.7% hexose, 1.8% hexosamine, and no hexuronic acid. This protein is rich in glycine, glutamic acid, and alanine, and contains neither hydroxyproline nor hydroxylysine. Sedimentation analysis showed a single peak with 1.8 S and the molecular weight was approx. 43,000 as determided by SDS polyacrylamide gel electrophoresis.

Aging and Hydroxyproline Content in Human Heart Muscle

Hydroxproline content was chemically determined in 45 human hearts obtained at autopsy, in order to know possible correlations between aging and hydroxyproline content in the heart muscle. There were no significant differences in the mean total hydroxyproline content between the epicardium and the endocardium, between left and right ventricles, nor between young, middle and old age groups. The hydroxyproline content in NaCl-soluble fraction decreased, and that in TCA-soluble fraction and in residue fraction increased with age. There were significant differences in the mean hydroxyproline content in NaCl-soluble fraction between middle and old age groups, and in residue fraction between young and old age groups in the endocardium of the left ventricle. Accordingly, the ratio of the content in NaCl-soluble fraction to that in TCA-soluble fraction decreases with age. This tendency was more evident in the epicardium than in the endocardium on the left ventricle. These results indicate that total hydroxyproline content per 1 mg of lyophilized material does not show any quantitative changes, but does show qualitative changes with age.

Collagen Content of Lungs of Mice after X-Ray Irradiation

To investigate the development of late radiation fibrosis in lungs large single doses of X rays were given to the hemithoraxes of mice. Hydroxyproline, an amino acid specific to collagen, was estimated in both irradiated and shielded lungs up to 48 weeks after exposure. There was a dose dependent atrophy of the irradiated lung and a compensatory hypertrophy of the shielded lung. The hydroxyproline per gram of dry irradiated lung increased between 24 and 36 weeks after 2000-4000 rad with no further change up to 48 weeks. Both total hydroxyproline content and dry weight of irradiated lungs decreased but after 24 weeks the histological appearance was that of collagen deposition after edema and hyperplasia. It is concluded that collagen deposition occurs at a late stage in the reorganization after radiation injury to mouse lung and is therefore probably not the cause of 80-180 day death after exposure of the whole thorax.

TREATMENT OF OSTEOPOROSIS OF AGEING WITH 1α-HYDROXYCHOLECALCIFEROL

Seven patients with osteoporosis of ageing were treated with synthetic 1α-hy- droxycholecalciferol (1α-H.C.C.) for 3-4 months. The compound was given at a daily oral dose of 2 μg together with an oral supplement of 1 g of calcium. Clinically there was a striking improvement in the patients' physical fitness. Increased bone formation and mineralisation were seen on iliac-crest bone biopsy, and this was supported by an increased osteoblastic activity demonstrated by histochemical measurement of alkaline-phosphatase activity. Bone histology furthermore showed a reduced bone resorption, which was supported by a reduced urinary excretion of total hydroxyproline. Photon absorptiometry of the forearm accorded with the histological findings, showing a significant increase in the bone mineral content. Serum-calcium rose in all patients, one developing a severe transitory hypercalcæmia. The urinary excretion of calcium and magnesium increased significantly. The serum concentrations of 25-hydroxycholecalciferol and parathyroid hormone were not significantly affected by the treatment. It is concluded that 1α-H.C.C. is an effective tool in the treatment of senile osteoporosis.

Calcium Kinetics in the Solanum malacoxylon-treated Rat

The chronic ingestion of the leaves of the plant Solanum malacoxylon (SM) causes an endemic disease in the cattle of some areas of Buenos Aires province. The animals affected manifest loss of appetite and weight, hypercalcemia, hyperphosphatemia, and ectopic calcifications. In order to study the mechanism of the hypercalcemia provoked by the administration of SM, a calcium kinetic study was performed in control and treated adult intact rats. The animals receiving SM showed higher levels of serum calcium throughout the study. The body excretion of 47Ca and the size of the most rapidly exchangeable calcium pool were also elevated. On the other hand, the bone accretion rate and the urinary excretion of total hydroxyproline were significantly diminished. The results indicate that during the early phase of SM administration in intact rats, bone turnover rate is depressed.

A simplified method for the estimation of urinary total hydroxyproline

A simplified method for the estimation of urinary total hydroxyproline

Serum alkaline phosphatase and urinary hydroxyproline values in children receiving phenobarbital with and without vitamin D

Concentrations of serum alkaline phosphatase and total urinary hydroxyproline were measured in 36 children to study the effect of phenobarbital administration with respect to the development of rickets in patients receiving anticonvulsive medications over prolonged periods of time. Administration of phenobarbital led to the appearance of increased AP and HOP values very early in the course of treatment and without any obvious bone changes suggestive of rickets; a single large oral dose of vitamin D had no appreciable effects in restoring the biochemical derangement. On the other hand, the administration of vitamin D in a daily dose of 4,000 IU for a period of two months hampered the appearance, or restored already existing changes of latent rickets, in children receiving anticonvulsive medication. The results in the present study favor the concept that phenobarbital administration is implicated in the development of rickets. The need for simultaneous daily administration of supplements of vitamin D in subjects receiving anticonvulsive drugs is stressed.

Effect of cellulose phosphate on calcium and magnesium homeostasis: studies in normal subjects and patients with latent hypoparathyroidism.

1. The bivalent cation-binding agent, cellulose phosphate, was given for 6 days to four normal subjects and six patients with latent hypoparathyroidism (diagnosed by impaired response to EDTA infusion), all of whom were on a moderately low calcium diet. 2. In normal subjects, there was a prompt and sustained fall in urinary calcium with no change in plasma calcium, indicating increased tubular reabsorption. Plasma and urinary magnesium fell, without increase in tubular reabsorption. The urinary total hydroxyproline increased and Tm,P/glomerular filtration rate fell after 2 days; these changes were transient and were consistent with a transient increase in parathyroid hormone secretion. 3. In the hypoparathyroid patients, urinary calcium fell more slowly and a fall in plasma calcium occurred in several subjects, the extent and duration of which corresponded with parathyroid status determined by EDTA infusion. Urinary conservation of calcium was impaired but plasma and urinary magnesium fell as in normal subjects. Urinary total hydroxyproline did not change and Tm,P/glomerular filtration rate fell more slowly than in the normal subjects. 4. The relative contributions of increased tubular reabsorption and reduced filtered load to calcium conservation in response to calcium depletion depend on the prevailing level of parathyroid function; the former is more important when parathyroid function is normal, the latter when parathyroid function is impaired. 5. In the detection of reduced parathyroid reserve, the assessment based on the plasma calcium response to cellulose phosphate agrees closely with the assessment based on the degree of recovery from EDTA-induced hypocalcaemia.

Determination of hydroxyproline polypeptides in urine and blood serum by gel filtration

Hydroxyproline-containing polypeptides were isolated from urine by filtration on polyacrylamide gel Bio-Gel P-2. This fraction was quantitatively and qualitatively similar to the nondialysable hydroxyproline in urines of normal adult subjects, patients with chronic uremia, and with primary or secondary hyperparathyroidism. Hydroxyproline polypeptides isolated from urines of patients with extensive Paget's disease or from normal growing subjects contained a group of smaller polypeptides slightly retarded on Bio-Gel P-2 which were not retained by the dialysis membrane Spectrapor™. In these two groups of subjects the values of hydroxyproline polypeptides were 35–45 percent higher than those of dialysable hydroxyproline. Increased excretion of hydroxyproline polypeptides was found in chronic uremia, primary and secondary hyperparathyroidism, Paget's bone disease and in normal growing subjects. With the notable exception of chronic uremia the excretion of hydroxyproline polypeptides paralleled that of total and oligopeptidic hydroxyproline. In some cases of chronic uremia the polypeptidic hydroxyproline represented the predominant fraction. On long columns of Bio-Gel P-6 hydroxyproline polypeptides separated into several peaks. The chromatographic pattern was rather characteristic for chronic uremia, secondary hyperparathyroidism, Paget's bone disease and physiological growth. A hydroxyproline fraction of similar Chromatographic properties was isolated from blood serum after deproteinisation with heat or ethanol at pH 4.8. High serum levels of this hydroxyproline fraction were observed in pathological conditions with increased urinary excretion of polypeptidic and nondialysable hydroxyproline.